Neisseria gonorrhoeae (Ng) is a prototypic stealth pathogen that uses multiple mechanisms to evade human immune defenses; consequently re-infection is a problem. This has frustrated attempts at vaccine development, while resistance to antimicrobials has made effective therapy more difficult and costly. New approaches are necessary to identify means to control this important pathogen, which causes almost 65 million cases on a worldwide basis, the majority in indigent populations and in less well-developed countries. Studies in our laboratory have demonstrated that Ng has the ability to form biofilms on the cervical epithelial cell surface. As part of these studies, we have been examining the role of gonococcal luxS in biofilm formation and have evidence that the gonococcus has a functional quorum sensing system that it uses to modify gene expression during biofilm formation. Furthermore, our studies have shown that the Ng autoinducer -2 (AI-2) interacts with the two component regulatory genes, misRS. Among the approximately 80 genes under the control of misRS are the gonococcal thermonuclease that is critical to remodeling the biofilm DNA matrix, and genes involved in peptidoglycan biosynthesis and cell division. We also observed that a homolog of a SIR-2 deacetylase and a novel acetyltransferase are also regulated by misRS, and based on these observations, we undertook proteomic studies of N-lysine acetylation and we discovered that N-lysine acetylation increases significantly in a Ng luxS mutant. A number of studies have shown that bacterial quorum agents can impact on signaling in eukaryotic cells. Our previous studies with Staphylococcus aureus have shown that within the confines of the phagolysome, autoinducer levels are sufficiently high to cause modifications in gene expression through the agr system and promote expression of virulence factors integral to survival in phagocytes. N. gonorrhoeae can survive within the phagolysome and induces an anti-apoptotic event in neutrophils. The gonococcus only has an AI-2 system lacking the enzymes to produce acylhomoserine lactone. Our interest is to examine whether the AI-2 produced by N. gonorrhoeae and N-lysine acetylation might play a role in pathogenesis after phagocytosis. The hypothesis that we will test in this proposal is that an intact Ng quorum sensing system functions within the PMN phagolysome and is necessary for survival of Ng in that environment. We will resolve this hypothesis by the following specific aims: Specific Aim 1: Determine the state of activation of the quorum sensing system of Ng ingested by human PMN and Specific Aim 2: Determine if and how mutations in the gonococcal quorum sensing system affect both the survival of Ng in PMN and the cell biology of Ng-laden PMN.